1. Field of the Invention
This invention relates to devices and methods for assaying antigens in a fluid sample. More particularly, it relates to a light-emitting assay for antigens having one binding site for antibodies.
2. Description of the Prior Art
Assays directed to detection and quantification of physiologically significant materials in biological fluid and tissue samples are important tools in scientific research and in the health care field. Immunoassays detect the reaction between immunological pairs, antibodies (Ab) and antigens (Ag), as set forth below: EQU Ag+Ab.fwdarw.Ag.multidot.Ab
Antibodies and antigens have complementary surfaces or cavities which specifically bind to a particular spatial and polar organization of the other molecule. The binding is reversible and antibodies and antigens may compete for limited binding sites. In typical assays, either the antigen, antibody or both may be labelled with an indicator molecule. The analyte of interest is most often the antigen; however, either antigen or antibody may be the object of the assay.
Several different types of assays have been developed which are capable of detecting relatively concentrated components of common biological samples such as human serum. Such assays include high-resolution agarose gel electrophoresis and test procedures based on the catalytic activity of endogenous enzymes. These methods generally do not have the sensitivity required to detect and quantify the numerous other physiologically important sample constituents which may be present at very low concentrations. Important constituents include endogenous molecules intimately involved in cellular regulation (hormones, steroids, biochemical messengers); basic structural components of the organism (amino acids, proteins, polysaccharides); genetic material (DNA, RNA); vitamins, drugs and drug metabolites; toxins, pathogens and substances generated by the immune system.
The early biological assay techniques for the clinically important serum constituents, such as the immunoprecipitation and immunodiffusion techniques developed in the 1940s, also lacked the sensitivity necessary to detect and quantify most serum constituents of medical interest. In 1956, Berson and Yalow reported detection of soluble insulin-antibody complexes in the serum of insulin-treated diabetics injected with radiolabelled preparations of the serum hormone. (See Berson et al., J. Clin. Invest. 35, 170 (1956).) The principles of this assay, commonly referred to as radioimmunoassay (RIA), were subsequently established and by the late 1960s the RIA was a major tool in endocrine laboratories. For example, virtually all the information now known about peptide hormonal physiology has resulted from the introduction of RIA and its ability to detect 10.sup.-10 to 10.sup.-12 molar concentrations of hormones.
The RIA assay technique was subsequently shown to be applicable to quantitative detection of any substance for which a specific antibody can be prepared, permitting development of a host of RIAs for chemical compounds such as drugs. In a broader sense, the RIA principle has also been extended to systems in which other binding substances replace antibody, for example, in receptor assays. In 1980, the sales of immunodiagnostic reagents alone were estimated to be $229 million.
Although exhibiting the desired sensitivity, RIAs have several disadvantages inherent in the required reagents. The use of radioactive isotopes requires a special permit and a special laboratory. For this reason, RIAs are performed by personnel separate from those in the routine clinical chemistry laboratory. Radiation can cause health hazards particularly for those working with the commonly used isotopes of iodine. In addition, the useful lifetime of the radiolabelled reagents employed is limited by half-life of the isotopes and the destructive processes that occur during isotopic decay. The equipment used to determine the amount of radioactivity in the samples is expensive and the counting of a series of samples is relatively time-consuming. (See Smith et al., Amer. Clin. Biochem. 18, 253-74 (1981).) Overall, the amount of automation in the immunodiagnostic area is much less than that found in the routine clinical laboratory. Using an eight-hour polyethylene glycol accelerated second antibody RIA for separation of free from bound antigen, only about 75-90 assays can be performed daily by a single technician employing manual pipettes and a single-channel gamma counter.
To overcome the problems associated with RIA, immunoassay techniques employing nonisotopic labels have been developed. These nonisotopic assays, referred to as enzyme-linked immunoabsorbent assays (ELISA), fluoroimmunoassays (FIA), and luminescent immunoassays (LIA), depending upon the label employed, avoid many of the problems associated with RIAs and possess sensitivities near to that of the RIA. More recently, enzyme-linked assays have become increasingly popular and are replacing RIAs in many cases due to their more simple protocol relative to RIAs. As many as 2,000 assays per day can be run by a technician employing a solid-phase ELISA in microtiter plates with manual pipettes. These types of assays also have permitted the development of "homogeneous" immunoassays in which the bound and free labelled material need not be separated prior to the detection and measurement step. The RIA procedure requires the separation of free from bound labelled material for estimation of the target substance concentration, a "heterogeneous" system. Sensitive assays in which antigen-antibody reactions could be detected without separation of free from complexed antigen are also more simple to automate.
A typical nonisotopic heterogeneous assay procedure in which the analyte is a member of an immunological pair may include contacting an immobilized reagent member of an immunological pair specific to the analyte with a sample potentially containing the analyte allowing the sample analyte member to bind with the reagent member. The unbound sample members are then physically separated from the immobilized reagent members. If the sample analyte members are labelled with a detectable agent, or if the reagent members have labelled opposite reagent members competing for the same binding sites as the analyte member, the concentration of analyte can be determined by an increase or decrease in the detection of the label agent.
A typical nonisotopic homogeneous assay may include reagent including both members of the immunological pair. The reagent immunological pairs interact to produce a detectable signal. The introduction of a sample containing the analyte member of the immunological pair causes a modulation of the signal produced by the reagent immunological pairs. The change in the signal corresponds to the concentration of the analyte member in the sample.
Although superior to RIAs in several respects, the nonisotopic assays described above also exhibit problems caused by endogenous interfering factors present in the reaction mixture. Proteins and other components commonly found in serum samples may exhibit fluorescent, chemiluminescent and enzymatic activity similar to that of the employed label. In addition, the activity of these labels may be inhibited by the presence of endogenous compounds which absorb or scatter the emitted light of photophore labels, similarly colored compounds relative to chromaphore labels, and catalytic enzymes which degrade enzyme labels. Determination of the activity of the employed label may also be impaired by the turbidity of the sample as in the case of whole blood samples. To a certain extent, these problems may be minimized by assay techniques employing a separation step wherein bound labelled material is separated from the sample, washed with buffer and the label activity is thereafter determined, or wherein the separation of bound and unbound labelled material is achieved by partitioning them between immiscible aqueous phases. See Mattiasson et al., Advances in Applied Microbiology 28, 117-47 (1981) and U.S. Pat. No. 4,312,944.
However, there exists in the art a need for a sample device and method for assaying small antigens and/or antibodies by nonisotopic means, particularly chemiluminescence which is adaptable to automated procedures and which will function in turbid samples without separation steps.